| Photodynamic therapy (PDT) is a novel and promising antitumor treatment, which is based on the tumor-specific accumulation of photosensitizing drugs that induce cancer cells death by yielding reactive oxygen species (ROS) after irradiation of light with an appropriate wavelength. Compared with traditional therapies, the outstanding advantage of PDT is that it destroys tumor through selective uptake of photosensitisers, and does not harm the normal surrounding tissues seriously.Photosensitisers play a key role in PDT. Because nanosized TiO2 photosensitiser in the presence of ultraviolet light can significantly inhibit tumor cells by yielding reactive oxygen species, and exhibits little toxicity, it is one of the potential promising antitumor photosensitisers. Nowadays, studies on photodynamic antitumor of nanosized TiO2 photosensitiser have become an active research field. These research mainly focused on antitumor of nanosized TiO2 photosensitiser prepared by gas phase, sol-gel and modification method in the presence of ultraviolet light. However, photodynamic inhibition mechanism of nanosized TiO2 photosensitiser prepared by hydrothermal method in liver cancer Bel-7402 cells has not been clarified.Because phthalocyanine compounds containing a planar macrocycle with an 18π-electron system in the presence of red light can significantly inhibit tumor cells by yielding reactive oxygen species, hydrophilic group redounds to the transport of drug in the body and lipophilic group conduces to the uptake of drug in tumor cells, phthalocyanine compounds with hydrophilic/lipophilic structure may become promising candidates for targeting photosensitisers. Nowadays, photodynamic antitumor of amphiphilic phthalocyanine photosensitisers have become an active research field. However, photodynamic inhibition mechanism of new amphiphilic tetra-α-(p-carboxyphenoxy) phthalocyanine zinc [TαPcZn] photosensitiser in liver cancer Bel-7402 cells has not been clarified.Taken together, photodynamic inhibition mechanism of nanosized TiO2 and amphiphilic TαPcZn photosensitisers in liver cancer Bel-7402 cells are promising and valuable. The main contents are as follows:1. Preparation of nanosized TiO2 photosensitiser and its photodynamic inhibiton in liver cancer cells1.1 Preparation and characterization of nanosized TiO2 photosensitiserNanosized TiO2 photosensitiserwas synthesized by hydrothermal method using titanium isopropoxide as raw materials. The as-prepared sample was studied by transmission electron microscope (TEM), X-ray diffraction (XRD),infrared (IR) spectrum and ultraviolet-visible (UV-vis) spectrum.1.2 Ultraviolet-light-excited nanosized TiO2 photosensitiser inhibits the proliferation of Bel-7402 cells through apoptosis inductionTo explore whether ultraviolet-light-excited nanosized TiO2 photosensitiser inhibits the proliferation of Bel-7402 cells by apoptosis induction, the effects of ultraviolet-light-excited nanosized TiO2 photosensitiser on proliferation and apoptosis were investigated by MTT, flow cytometry analysis of Annexin V-FITC/PI double labeling and HE staining assay. MTT assay showed that ultraviolet-light-excited nanosized TiO2 photosensitiser apparently inhibited the proliferation of Bel-7402cancer cells. Flow cytometry analysis of Annexin V-FITC/PI double labeling assay showed that ultraviolet-light-excited nanosized TiO2 photosensitiser apparently induced the apoptosis of Bel-7402 cells. HE staining assay showed that Bel-7402 cells treated by ultraviolet-light-excited nanosized TiO2 photosensitiser apparently exhibited morphological characteristics of apoptosis and necrosis. Taken together, ultraviolet-light-excited nanosized TiO2 photosensitiser inhibits the proliferation of Bel-7402 cells by apoptosis induction.1.3 Effects of ultraviolet-light-excited nanosized TiO2 photosensitiser on Bcl-2 and caspase-3 in Bel-7402 cellsTo explore whether Bcl-2 and caspase-3 are involved in the apoptosis of Bel-7402 cells induced by ultraviolet-light-excited nanosized TiO2 photosensitiser, effects of ultraviolet-light-excited nanosized TiO2 on Bcl-2 and caspase-3 were assayed by western blotting method. The results showed that ultraviolet-light-excited nanosized TiO2 photosensitiser led to down-regulation of Bcl-2 and activation of caspaes-3, suggesting that down-regulation of Bcl-2 and activation of caspaes-3 are involved in the apoptosis of Bel-7402 cells induced by ultraviolet-light-excited nanosized TiO2 photosensitiser. In other word, ultraviolet-light-excited nanosized TiO2 induces Bel-7402 cells apoptosis by down-regulation of Bcl-2 and activation of caspaes-3.2. Preparation of TαPcZn photosensitiser and its photodynamic inhibition in liver cancer cells2.1 Preparation and characterization of TαPcZn photosensitiserTa PcZn photosensitiser was synthesized by template reaction method using 3-[p-(carboxyphenoxy) phthalonitrile as raw materials. The as-prepared sample was studied by elemental analysis, laser desorption/ionization time off light mass spectrometry (LDI-TOF-MS), hydrogen nuclear magnetic resonance spectrum (1HNMR), IR spectrum, UV-vis spectrum and fluorescene spectrum. Composition and structure of TαPcZn photosensitiser are determined.2.2 Photostability of TαPcZnPhotostability of T a PcZn in acetone was assayed by spectrophotometer. The results showed that auto-sensitized photooxidation led to decomposition of TαPcZn in the presence of ultraviolet light, and the process obeyed first order reaction rule.2.3 Red-light-excited TαPcZn photosensitiser inhibits the proliferation of Bel-7402 cells through apoptosis inductionTo explore whether red-light-excited TαPcZn photosensitiser inhibits the proliferation of Bel-7402 cells by apoptosis induction, effects of red-light-excited TαPcZn photosensitiser on proliferation and apoptosis were investigated by MTT, flow cytometry analysis of Annexin V-FITC/PI double labeling, inverted microscope and transmission electron microscopy assay. MTT assay showed that red-light-excited TαPcZn photosensitiser apparently inhibited the proliferation of Bel-7402 cells. Flow cytometry analysis of Annexin V-FITC/PI double labeling assay showed that red-light-excited TαPcZn photosensitiser apparently induced the apoptosis of Bel-7402 cells. Inverted microscope and transmission electron microscopy assay showed that Bel-7402 cancer cells treated by red-light-excited TαPcZn photosensitiser apparently exhibited morphological characteristics of apoptosis and necrosis. Taken together, red-light-excited TαPcZn photosensitiser inhibits the proliferation of Bel-7402 cells by apoptosis induction.2.4 Red-light-excited TαPcZn photosensitiser inhibits the proliferation of Bel-7402 cells through cycle arrestTo explore whether red-light-excited TαPcZn photosensitiser inhibits the proliferation of Bel-7402 cancer cells by cycle arrest, effects of red-light-excited TαPcZn photosensitiser on cell cycle were investigated by flow cytometry analysis of Annexin PI labeling assay. The results showed that red-light-excited TαPcZn photosensitiser apparently resulted in a decrease in the percentage of cells in G0/G1 and G2/M phase, and an increase in the percentage of cells in S phase, suggesting that red-light-excited TαPcZn photosensitiser inhibits the proliferation of Bel-7402 cells through S cell cycle arrest.2.5 Effect of red-light-excited TαPcZn photosensitiser on Bcl-2 in Bel-7402 cellsTo explore whether Bcl-2 is involved in apoptosis of Bel-7402 cells induced by red-light-excited TαPcZn photosensitiser, effect of red-light-excited TαPcZn on Bcl-2 was assayed by western blotting method. The results showed that red-light-excited TαPcZn photosensitiser led to down-regulation of Bcl-2, suggesting that down-regulation of Bcl-2 is involved in the apoptosis of Bel-7402 cells induced by red-light-excited TαPcZn photosensitiser. In other word, red-light-excited TαPcZn photosensitiser induces Bel-7402 cells apoptosis by down-regulation of Bcl-2.
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